Project Summary/Abstract The pathogenesis of dermatomyositis (DM), an autoimmune disease predominantly affecting skin and muscle, is poorly understood. Increased levels of IFN? have been reported in DM patients. Levels of IFN? correlate with Cutaneous Dermatomyositis Disease Area and Severity Index (CDASI) activity. IFN?, but not IFN?, drives the disease activity in DM. Mitochondrial antiviral-signaling protein (MAVS) is an intracellular adaptor protein linking cytoplasmic RNA sensors, retinoic acid-inducible gene-I (RIG-I), which trigger the production of IFN?. The homotypic interaction between the CARDs of MAVS and the CARDs of RIG-I form protein aggregates and functional clusters on the surface of mitochondria. The MAVS aggregates activate NF- ?B and IRF3, then subsequently resulting in IFN? production. Peripheral blood mononuclear cells (PBMCs) from SLE patients contain MAVS in spontaneous aggregation that correlates with the increased levels of IFN?. Recent studies indicate that MAVS can also be detected extracellularly in the plasma of lupus patients, and in the conditioned medium of cultured cells. The extracellular MAVS, isolated with anti-MAVS conjugated beads from lupus plasma, can be taken up by dendritic cells (DC) and result in IFN? production. The underlying mechanisms are unclear. Extracellular vesicles (EVs) harbor bioactive molecules, and mediate intercellular communications. Studies from our and other groups indicate the importance of EVs in disease pathogenesis, and that EVs can also serve as a biomarker for monitoring of disease severity. Our preliminary studies showed that the extracellular MAVS associated with EVs in an aggregated form, and EVs can mediate the delivery of these MAVS aggregates into the recipient cells for functional activation of the downstream effector NF-kB and production of IFN? in myeloid dendritic cells (mDCs). Several studies have reported the increased plasma levels of EVs in patients with DM. Our preliminary data also showed the existence of MAVS aggregates in the plasma of DM, and the isolated EVs from plasma of DM patients can activate the downstream effector NF-kB in mDCs. Therefore, our central hypothesis is that EV-associated extracellular MAVS can functionally enhance type I IFN production and propagate inflammatory responses, therefore playing an important role in DM. In Aim 1, we will investigate the cellular mechanisms of EV-associated MAVS in IFN? induction in mDCs by using MAVS deficient and wildtype cells. In Aim 2, we will determine the role of EV-associated MAVS in IFN? production, and their relationship with the disease activity in patients with DM. Overall, the proposed studies will investigate the cellular mechanisms of EV-associated extracellular MAVS in IFN? production, and the potential involvement of MAVS-positive EVs in autoimmune skin inflammation in DM. This study will advance our understanding of the disease pathophysiology and provide insights into new therapeutic strategies in the future.